 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 25, 2021 |
| Title |
cel-miR-67 |
| Sample type |
SRA |
| |
|
| Source name |
Cardiomyocyte
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Wistar
tissue: heart
age: post natal day 1
genotype: wild type
cell type: ventricular cardiomyocytes
treatment: transfected with cel-miR-67
|
| Treatment protocol |
Ventricular CMs were isolated from neonatal rat. Briefly, ventricles from neonatal rat (day 0) were separated from the atria, cut into pieces and then dissociated in calcium and bicarbonate-free Hanks with HEPES (CBFHH) buffer containing 1.75 mg/ml trypsin (BD Difco) and 10 mg/ml DNase II (Sigma), using a mechanical and physical stirring. Digestion was performed at room temperature in ten-min steps with collection of the surnatant to fetal bovine serum (FBS, Life Technologies) after each step. The surnatant was collected and centrifuged to pellet the cardiomyocytes, which were then resuspended in Dulbecco's modified Eagle medium 4.5 g/l glucose (DMEM, Life Technologies) supplemented with 5% FBS, 20 mg/ml vitamin B12 (Sigma) and with 100 U/ml of penicillin and 100 mg/ml of streptomycin (Sigma). The collected cells were passed through a cell strainer (40mm, BD Falcon) and then plated onto 100-mm plastic dishes for 2 hr at 37C in 5% CO2 and humidified atmosphere. Then, the surnatant, which contains mostly cardiomyocytes, was collected and counted.The cells were plated in 60-cm primary dishes at the concentration of 1,5*10^6 cells.The miRNAs were obtained from Dharmacon, Thermo Scientific. After 24 hr miRNAs were transfected into neonatal rat CMs using a standard transfection protocol, at the final concentration of 25 nM. Briefly, the transfection reagent (Lipofectamine RNAiMAX, Life Technologies) was diluted in OPTI-MEM (Life Technologies) and added to the selected miRNAs; 30 min later, each mix, containing the transfection reagents and the miRNAs, was added to the Dulbecco's modified Eagle medium 4.5 g/l glucose (DMEM, Life Technologies) supplemented with 5% FBS and 20 mg/ml vitamin B12 (Sigma) in absence of antibiotics. Twenty-four hours after transfection, culture medium was replaced by fresh medium.
|
| Growth protocol |
Neonatal rat cardiomyocytes were grown in the Dulbecco's modified Eagle medium 4.5 g/l glucose (DMEM, Life Technologies) supplemented with 5% FBS, 20 mg/ml vitamin B12 (Sigma) and with 100 U/ml of penicillin and 100 mg/ml of streptomycin (Sigma) at 37°C in 5% CO2 and humidified atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cardiomyocyte cultures were isolated by enzymatic dissociation of 1 day-old neonatal rat hearts. Neonatal cardiomyocytes were seeded on Primaria 10 cm dishes and one day later, cells were transfected with mimics (Life Technologies) of hsa-miR-106b-5p, hsa-miR-93-5p, hsa-miR-25-3p or cel-miR-67 as control (25mM) using (Lipofectamine RNAiMAX, Life Technologies) and 72 hrs later RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit was used with 2 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Illumina Casava1.7 software used for basecalling.
The raw sequencing reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to Ensembl Rattus norvegicus reference genome (GCA_000001895.4 Rnor 6.0.89.6) using STAR 2.7 with default parameters.
Rounded gene counts were normalized to RPKM (reads per kilobase of exon model per million mapped reads) using the rpkm function in the Bioconductor package edgeR (McCarthy, J. D, Chen, Yunshun, Smyth, K. G (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research, 40(10)).
Genes with RPKM values greater than 2.00 in both miRNA and cel-miR-67 transfected rat CMs were considered as expressed genes.
Fold changes were taken with respect to the expression upon cel-miR-67 transfection (control condition). Genes whose expression fold change were greater than 1.3 were considered as differentially expressed.
Genome_build: Rnor 6.0.89.6
Supplementary_files_format_and_content: tab-delimited text files include RPKM values
|
| |
|
| Submission date |
Jun 24, 2021 |
| Last update date |
Jun 30, 2021 |
| Contact name |
Ryan John A Cubero |
| E-mail(s) |
[email protected]
|
| Organization name |
IST Austria
|
| Lab |
Lab Siegert
|
| Street address |
Am Campus 1
|
| City |
Klosterneuburg |
| State/province |
Lower Austria |
| ZIP/Postal code |
3400 |
| Country |
Austria |
| |
|
| Platform ID |
GPL14844 |
| Series (1) |
| GSE178867 |
RNA-seq profiles of rat cardiomyocytes overexpressing hsa-miR-106b-5p, hsa-miR-93-5p, hsa-miR-25-3p or cel-miR-67 |
|
| Relations |
| SRA |
SRX11222194 |
| BioSample |
SAMN19960791 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5399474_RPKM_miR67.txt.gz |
254.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
